1: Cancer. 2005 Oct 15;104(8):1678-86. Molecular classification of melanoma using real-time quantitative reverse transcriptase-polymerase chain reaction. Lewis TB, Robison JE, Bastien R, Milash B, Boucher K, Samlowski WE, Leachman SA, Dirk Noyes R, Wittwer CT, Perreard L, Bernard PS. Research and Development, ARUP Laboratories Inc., Salt Lake City, Utah. BACKGROUND: The early detection and characterization of metastatic melanoma are important for prognosis and management of the disease. Molecular methods are more sensitive in detecting occult lymph node metastases compared with standard histopathology and are reported to have utility in clinical diagnostics. METHODS: Using real-time quantitative reverse transcriptase-polymerase chain reaction ([q]RT-PCR), the authors examined 36 samples (30 melanomas, 4 benign nevi, and 2 reactive lymph nodes) for the expression of 20 melanoma-related genes that function in cell growth and differentiation (epidermal growth factor receptor [EGFR], WNT5A, BRAF, FOS, JUN, MATP, and TMP1), cell proliferation (KI-67, TOP2A, BUB1, BIRC5, and STK6), melanoma progression (CD63, MAGEA3, and GALGT), and melanin synthesis (TYR, MLANA, SILV, PAX3, and MITF). In addition, samples were tested for mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3). RESULTS: Hierarchical clustering analysis of the expression data was able to distinguish between the melanoma and nonmelanoma samples and further stratified the melanoma samples into two groups differentiated by high expression of the genes involved in beta-catenin activation (EGFR and WNT5A) and the MAPK/ERK pathway (BRAF, FOS, and JUN). Eighteen of the 28 patients (64%) were found to have mutations in either exon 15 of BRAF (V599 substitution) or codon 61 of NRAS. The mutations were mutually exclusive and did not appear to be associated with the different expression subtypes. CONCLUSIONS: The results of the current study demonstrate that real-time qRT-PCR can be analyzed using hierarchical clustering to identify expression patterns that differentiate between melanomas and other tissue types. Using a supervised analysis of the data, the authors found that the best discriminators for molecularly distinguishing between melanoma, benign nevi, and lymph nodes were MLANA, CD63, and BUB1. These markers could have diagnostic utility for the detection of melanoma micrometastasis in sentinel lymph nodes. Cancer 2005. (c) 2005 American Cancer Society. PMID: 16116595 [PubMed - in process] --------------------------------------------------------------- 2: J Cell Sci. 2002 Feb 1;115(Pt 3):531-41. Pax3 regulates morphogenetic cell behavior in vitro coincident with activation of a PCP/non-canonical Wnt-signaling cascade. Wiggan O, Hamel PA. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada. Mutations to Pax3 and other Pax family genes in both mice and humans result in numerous tissue-specific morphological defects. Little is known, however, about the cellular and molecular mechanisms by which Pax genes regulate morphogenesis. We previously showed that Pax3 induces cell aggregation and a mesenchymal-to-epithelial transition in Saos-2 cells. We show here that Pax3-induced aggregates arise through the formation of distinct structures involving cell rearrangements and cell behaviors resembling those that occur during gastrulation and neurulation known as convergent extension. During these Pax3-induced processes, Dishevelled and Frizzled are localized to the actin cytoskeleton and both proteins coimmunoprecipitate focal adhesion components from detergent-insoluble cell fractions. We show further that these Pax3-induced cell movements are associated with activation of a Wnt-signaling cascade, resulting in induction and activation of c-Jun-N-terminal kinase/stress activated protein kinase (JNK/SAPK). All of these Wnt-signaling factors exhibit altered subcellular distribution in Pax3-expressing cells. In particular, we show the localization of JNK/SAPK to both the nucleus and to cytoplasmic multi-vesicular structures. These data show that Pax3 regulates morphogenetic cell behavior and that regulation of a conserved, planar cell polarity/noncanonical Wnt-signaling cascade entailing JNK activation is a function of Pax3 activity. PMID: 11861760 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Epilepsia. 1997 Apr;38(4):415-23. Phenytoin-induced teratogenesis: a molecular basis for the observed developmental delay during neurulation. Bennett GD, Lau F, Calvin JA, Finnell RH. Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station 77843-4458, USA. PURPOSE: We wished to determine whether chronic phenytoin (PHT) exposure could impair neural development and if any morphological alterations could be linked to changes in gene expression. METHODS: Pregnant SWV mice were chronically administered PHT 40 mg/kg/day from gestational day (GD) 0:12 (day:h) until they were killed at various timepoints throughout neural tube closure (NTC). At each timepoint, embryos from both treated and control dams were collected and scored for their progression through NTC. The neural tubes were then isolated and subjected to in situ transcription (IST) and antisense RNA amplification procedures. Using these techniques, we examined the expression of 10 genes: N-cadherin (Ncad), collagen type IV (col-IV), bcl-2, c-jun, PAX-3, collular retinol binding protein-2 (CRBP-2), retinoic acid receptor alpha (RAR alpha), transforming growth factor(beta2) (TGF(beta2)), wee-1, and EMX-2. RESULTS: Chronic PHT exposure not only caused a delay in NTC whereby exposed embryos lagged behind the controls at each collection timepoint, but also significantly altered the expression of specific genes at distinct times during NTC. Early in NTC, PHT induced a significant reduction in the expression of N-cad, col-IV, and c-jun in exposed embryos as compared with controls. In contrast, during the midstages of NTC, the only significant molecular alterations observed in the PHT-exposed embryos was the continued decreased expression of col-IV and an increase in CRBP-2 expression. Finally, in the latter stages of NTC, PHT caused a significant reduction in the expression of bcl-2, RAR alpha, TGF(beta2), EMX-2, and PAX-3. CONCLUSIONS: These results show that although the effects of PHT are morphologically subtle, causing a delay in the development of the neural tube, this delay is accompanied by alterations in critical genes at crucial times of neural development that may account for the observed neurological deficits often associated with PHT exposure. PMID: 9118846 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------